Fig 2: Relative total protein levels of RAGE andligands S100B and HMGB1 in white adipose tissue (WAT) of 13-lined groundsquirrels.(A) Histogram showing relative mean protein levels of RAGE, S100B, and HMGB1 (± S.E.M., n = 4 independent protein isolations from different animals). (B) Representative western blots for certain torpor-arousal time points. A space between representative band images indicates a removal of other lanes from the same blot. Data were analyzed using ANOVA with Tukey’s post-hoc test. Shared letters indicate data are not significantly different from each other and different letters indicate statistical significant differences between sample points (p < 0.05).

Fig 3: AGE-RAGE signaling can activate ERK1/2 signaling and downstream pro-inflammatory transcription factors.The advanced glycation end-product (AGE)—AGE receptor (RAGE) pathway can be activated by the binding of damage-associated molecular pattern molecules (DAMPs) like S100B (S100 calcium-binding protein B) and HMGB1 (high mobility group box 1 protein) from dying cells, or AGEs, derived from oxidized proteins, lipids, and nucleic acids. RAGE can signal through the MAPK (Mitogen-activated protein kinase) signaling transduction proteins like Raf (MAPK kinase kinase), MEK1 (MAPK kinase), ERK1/2 (Extracellular Signal-Regulated Kinase), leading to the phosphorylation and nuclear translocation of key transcription factors like Ets1, Jun and NFAT5 (Nuclear factor of activated T-cells 5).

Fig 4: Relative total protein levels of RAGE andligands S100B and HMGB1 in brown adipose tissue (BAT) of 13-lined groundsquirrels.(A) Histogram showing relative mean protein levels of RAGE, S100B, and HMGB1 (± S.E.M., n = 4 independent protein isolations from different animals). (B) Representative western blots for certain torpor-arousal time points. A space between representative band images indicates a removal of other lanes from the same blot. Data were analyzed using ANOVA with Tukey’s post-hoc test. Shared letters indicate data that are not significantly different from each other and different letters indicate statistical significant differences between sample points (p < 0.05).

Fig 5: Expression of S100 calcium-binding protein B (S100B) and phosphorylation of C-Jun N-terminal kinase (p-JNK) in the ipsilateral cortex were significantly improved with H2 gas inhalation despite hypoperfusion. (A) Representative bands of S100B and p-JNK in the left temporal cortex together with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeping protein (n = 5, representatively). (B,C) Quantification of S100B/GAPDH (B) and p-JNK/GAPDH (C). *P < 0.05. D: Representative images of immunofluorescence double staining of p-JNK (red) with neuronal nuclear antigen (NeuN), Iba1, or glial fibrillary acidic protein (GFAP) (green) in the left temporal cortex in the control group and H2 group. Nuclei are stained blue with 4',6-diamidino-2-phenylindole. Scale bar = 20 µm.